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Molecular biology
Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and learning how these interactions are regulated. Writing in Nature, William Astbury described molecular biology as : "... not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned particularly with the forms of biological molecules and ..... is predominantly three-dimensional and structural - which does not mean, however, that it is merely a refinement of morphology - it must at the same time inquire into genesis and function" Relationship to other "molecular-scale" biological sciences Researchers in molecular biology use specific techniques native to molecular biology (see Techniques section later in article), but increasingly combine these with techniques and ideas from genetics, biochemistry and biophysics. There is not a hard-line between these disciplines as there once was. The following figure is a schematic that depicts one possible view of the relationship between the fields: *''Biochemistry'' is the study of the chemical substances and vital processes occurring in living organisms. *''Genetics'' is the study of the effect of genetic differences on organisms. Often this can be inferred by the absence of a normal component (e.g. one gene). The study of "mutants" – organisms which lack one or more functional components with respect to the so-called "wild type" or normal phenotype. Genetic interactions such as epistasis can often confound simple interpretations of such "knock-out" studies. *''Molecular biology'' is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still provides a good starting point for understanding the field. This picture, however, is undergoing revision in light of emerging novel roles for RNA. Much of the work in molecular biology is quantitative, and recently much work has been done at the interface of molecular biology and computer science in bioinformatics and computational biology. As of the early 2000s, the study of gene structure and function, molecular genetics, has been amongst the most prominent sub-field of molecular biology. Increasingly many other fields of biology focus on molecules, either directly studying their interactions in their own right such as in cell biology and developmental biology, or indirectly, where the techniques of molecular biology are used to infer historical attributes of populations or species, as in fields in evolutionary biology such as population genetics and phylogenetics. There is also a long tradition of studying biomolecules "from the ground up" in biophysics. Techniques of molecular biology Since the late 1950s and early 1960s, molecular biologists have learned to characterise, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells. Expression cloning One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells is called transformation, and can be completed with several methods, including electroporation, microinjection, passive uptake and conjugation. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Several different transfection techniques are available, including calcium phosphate transfection, liposome transfection, and proprietary transfection reagents such as Fugene. DNA can also be introduced into cells using viruses or pathenogenic bacteria as carriers. In such cases, the technique is called viral/bacterial transduction, and the cells are said to be transduced. In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied. Polymerase chain reaction (PCR) Main article: Polymerase chain reaction The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. Gel electrophoresis Main article: Gel electrophoresis Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated using an electric field. In agarose gel electrophoresis, DNA and RNA can be separated based on size by running the DNA through an agarose gel. Proteins can be separated based on size using an SDS-PAGE gel. Proteins can also be separated based on their electric charge, using what is known as an isoelectric gel. Southern blotting Main article: Southern blot The Southern blot is a technique employed to ascertain information about the molecular weight and relative amount of a DNA sequence of interest. The assay was first developed by Edwin Southern and is a combination of gel electrophoresis of DNA (often first fragmented by restriction enzyme digestion), transfer of the same to a charged membrane, and hybridization of a labeled DNA probe. Following hybridization, the membrane is washed to remove unbound probe, and an image obtained via autoradiography or using equipment such as a phosphoimager. The image will indicate the location(s) to which the probe hybridized, with the intensity of the signal observed serving as a measure of relative abundance. Northern blotting Main article: Northern blot The Northern blot is used to study the expression patterns a specific type of RNA molecule as relative comparison among of a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used, however, most result in the revelation of bands representing the sized of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expressing is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determing at what time certain genes are expressed in living tissues. Western blotting and immunochemistry Main article: Western blot Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey. These antibodies can be used for a variety of analytical and preprative techniques. In Western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates. This technique is called SDS-PAGE (for Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including chemiluminescence or radioactivity. Antibodies can also be used to purify proteins. Antibodies to a protein are generated and are often then coupled to "beads". After the antibody has bound to the protein of interest, this antibody-protein complex can be separated from all other proteins by centrifugation. During centrifugation, the beads, to which the antibody is coupled, will pellet (bringing the protein of interest down with it) whereas all other proteins will remain in the solution. Alternatively, antibodies coupled to a solid support matrix like Sephadex or Sepharose beads, for example, can be used to remove a protein of interest from a complex solution. After washing unbound and non-specifically bound materials away from the "beads", the protein of interest is then eluted from the matrix, usually by adding a solution with a high salt concentration, or by varying the pH of the solution in which the matrix is contained. The beads can either be suspended in solution (batch processing) or packed into a tube (column processing). History Molecular biology was established in the 1930s, the term was first coined by Warren Weaver in 1938 however. Warren was director of Natural Sciences for the Rockefeller Foundation at the time and believed that biology was about to undergo a period of significant change given recent advances in fields such as X-ray crystallography. He therefore channeled significant amounts of (Rockefeller Institute) money into biological fields. Further reading * Keith Roberts, Martin Raff, Bruce Alberts, Peter Walter, Julian Lewis and Alexander Johnson, Molecular Biology of the Cell 4th Edition, Routledge, March, 2002, hardcover, 1616 pages, 7.6 pounds, ISBN 0815332181 3th Edition, Garland, 1994, ISBN 0815316208 2nd Edition, Garland, 1989, ISBN 0824036956 Notes * W.T. Astbury, [Nature 190, 1124 (1961)] See also * Cell biology (structures and components of the cell) * DNA and chromosome structure * Protein biosynthesis (transcription from DNA to RNA, translation from RNA into protein) * Protein structure and diversity * Genome * Important publications in molecular biology * List of molecular biology topics * Proteome Notable molecular biologists * Christiane Nüsslein-Volhard * Frederick Sanger * Francis Crick * Francois Jacob * James D. Watson * Max Perutz * Rosalind Franklin * Susumu Tonegawa In fiction and games * Genome soldiers (MGS) External links *BioDirectory: Search Engine for Biological Web Resources *Institute of Molecular and Cell Biology *''Nature Reviews Molecular Cell Biology'' (journal home) *Stanford Encyclopedia of Philosophy entry *The Virtual Library of Biochemistry and Cell Biology *Western Blot Protocol Resources *Research Highlights on reporter genes used in molecular biology References *Cohen, S.N., Chang, A.C.Y., Boyer, H. & Heling, R.B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70, 3240 – 3244 (1973). *Rodgers, M. The Pandora's box congress. Rolling Stone 189, 37 – 77 (1975). *